title: Time-lapse observation of the dedifferentiation process in mouse chondrocytes using chondrocyte-specific reporters.
abstract: OBJECTIVE
  When chondrocytes prepared from cartilage are expanded in monolayer culture, fibroblast-like cells gradually prevail. Although these prevailing fibroblast-like cells are believed to emerge because of the dedifferentiation of chondrocytes, the definite origin of the prevailing fibroblast-like cells has not been determined. We herein examined whether the prevailing non-chondrocytic cells observed after monolayer expansion culture arise from dedifferentiating chondrocytes or are the result of the overgrowth of fibroblasts that are present at the start of the culture. We also evaluated whether chondrocytes dedifferentiate because they proliferate or because they are cultured in monolayers.
  
  
  METHODS
  Chondrocytes were prepared from Col11a2-EGFP transgenic mice and Col11a2-Cre; R26-stop(flox)-EYFP transgenic mice, which respectively express enhanced green fluorescent protein (EGFP) and Cre specifically in chondrocytes under the control of Col11a2 promoter/enhancer sequences. Col11a2-Cre; R26-stop(flox)-EYFP mice express enhanced yellow fluorescent protein (EYFP) only in cells which express or used to express Cre. We performed a time-lapse observation of the chondrocytes during monolayer expansion culture, and also observed the chondrocytes after treatment with mitomycin C.
  
  
  RESULTS
  A time-lapse observation showed that Col11a2-EGFP chondrocytes underwent cell divisions, lost GFP fluorescence, increased cell numbers, and prevailed during the expansion culture. The observation of the Col11a2-Cre; R26-stop(flox)-EYFP chondrocytes confirmed that most of the cells after expansion in monolayer culture had been chondrocytes. Mitotically inactive chondrocytes generated by treatment with mitomycin C still underwent dedifferentiation, thus suggesting that chondrocyte dedifferentiation is not associated with cell division.
  
  
  CONCLUSIONS
  The non-chondrocytic cells that prevail after the monolayer expansion culture of chondrocytes originate from chondrocytes, and are not generated by the overgrowth of fibroblasts that are present at the start of the culture. Chondrocyte dedifferentiation does not appear to be associated with cell division.
authors.affiliation: Department of Cell Growth and Differentiation, Center for iPS Cell Research and Application, Kyoto University, 53 Kawahara-cho, Shogoin, Sakyo-ku, Kyoto 606-8507, Japan; Department of Plastic Surgery, Osaka University Graduate School of Medicine, 2-2 Yamadaoka, Suita, Osaka 565-0871, Japan.
authors.forename: Y
authors.initials: Y
authors.lastname: Minegishi
authors.forename: K
authors.initials: K
authors.lastname: Hosokawa
authors.forename: N
authors.initials: N
authors.lastname: Tsumaki
mesh_terms: D000818:Animals; D001426:Bacterial Proteins; D002358:Cartilage, Articular / Q000166:cytology* / Q000378:metabolism; D018929:Cell Culture Techniques; D054337:Cell Dedifferentiation; D002454:Cell Differentiation; D019902:Chondrocytes / Q000166:cytology* / Q000378:metabolism; D024161:Collagen Type XI / Q000235:genetics; D049452:Green Fluorescent Proteins; D008164:Luminescent Proteins; D051379:Mice; D008822:Mice, Transgenic; D061848:Optical Imaging; D059008:Time-Lapse Imaging
chemical_list: D001426:Bacterial Proteins; C497046:Col11a2 protein, mouse; D024161:Collagen Type XI; D008164:Luminescent Proteins; C485184:enhanced green fluorescent protein; C054562:yellow fluorescent protein, Bacteria; D049452:Green Fluorescent Proteins
keywords: Col11a2; Dedifferentiation; Time-lapse observation; Transgenic mice